University of Groningen
Competitive Binding of the SecA ATPase and Ribosomes to the SecYEG Translocon
Wu, Zht Cheng; de Keyzer, Jeanine; Kedrov, Alexej; Driessen, Arnold
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The Journal of Biological Chemistry
DOI:
10.1074/jbc.M111.297911
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Wu, Z. C., de Keyzer, J., Kedrov, A., & Driessen, A. J. M. (2012). Competitive Binding of the SecA ATPase
and Ribosomes to the SecYEG Translocon. The Journal of Biological Chemistry, 287(11), 7885-7895. DOI:
10.1074/jbc.M111.297911
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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 11, pp. 7885–7895, March 9, 2012
© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
Competitive Binding of the SecA ATPase and Ribosomes to
the SecYEG Translocon*□
S
Received for publication, August 25, 2011, and in revised form, January 11, 2012 Published, JBC Papers in Press, January 20, 2012, DOI 10.1074/jbc.M111.297911
Zht Cheng Wu, Jeanine de Keyzer, Alexej Kedrov, and Arnold J. M. Driessen1
From the Department of Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of
Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands
Background: Both SecA and the ribosome need to interact with the translocon during membrane protein insertion.
Results: SecA competes with ribosomes and ribosome-nascent chain complexes for binding to the translocon.
Conclusion: SecA and ribosome binding to the translocon is mutually exclusive, implying that during membrane protein
insertion, both ligands bind the translocon in a sequential manner.
Significance: Insight in the mechanism of membrane protein insertion.
In Escherichia coli the conserved heterotrimeric membrane
protein complex SecYEG (also termed translocon) mediates the
transport of secretory proteins across and the insertion of
membrane proteins into the cytoplasmic membrane (for
review, see Ref. 1). Secretory proteins are mostly targeted posttranslationally to the SecYEG complex, after which translocation is driven by the peripheral motor domain, the ATPase
SecA. Membrane proteins mostly follow a co-translational targeting route in which ribosome-nascent chain complexes
(RNCs)2 are directed to the SecYEG complex by signal recognition particle and its membrane-associated receptor FtsY (for
review, see Ref. 2)). After docking of the ribosome on SecYEG,
insertion occurs concomitantly with the elongation of the polypeptide chain on the ribosome, whereas translocation of large
periplasmic loops requires the assistance of SecA (3, 4).
* This work was supported by the Chemical Sciences division of the Netherlands Foundation for Scientific Research (CW-NWO).
This article contains supplemental Figs. S1–S4.
1
To whom correspondence should be addressed. Tel.: 31-50-3632164; Fax:
31-50-3632154; E-mail: [email protected].
2
The abbreviations used are: RNC, ribosome-bound nascent chain; SPR,
surface plasmon resonance; FCS, fluorescence correlation spectroscopy; IMV, inner membrane vesicles; TMS, transmembrane segment;
DDM, n-dodecyl ␤-D-maltoside; AMP-PNP, adenosine 5⬘-(␤,␥-imino)triphosphate; ND, nanodiscs.
□
S
MARCH 9, 2012 • VOLUME 287 • NUMBER 11
The initiation of protein translocation or membrane protein
insertion is dependent on the high affinity interactions between
the SecYEG complex and SecA or the ribosome. In recent years,
structural, biochemical, and computational approaches have
provided detailed insights in the interaction between SecYEG
and its cytosolic binding partners. Both SecA and the ribosome
interact primarily with the largest subunit of the SecYEG complex, i.e. SecY. This interaction involves multiple contact
points, and the main connections are formed by the ribosomal
23S rRNA, the ribosomal protein L23, and the fourth and fifth
cytoplasmic loops (C4 and C5) of SecY (5–9). Substitution of
the conserved arginine residues in these SecY loops severely
reduces the interaction with the ribosome (7). In addition, the
C-terminus of SecY interacts with the ribosomal protein L24,
and the N terminus and amphipathic helix of SecE interact with
proteins L23 and L29 (7–9). Intriguingly, SecA appears to bind
to the same SecY loops that are important for ribosome interaction. Indeed, a recent crystal structure of SecYEG in complex
with SecA (10) demonstrated contacts between the SecY C4
and C5 loops and SecA (11, 12). Mutational analysis identified
the arginine at position 357 in the C5 loop as indispensable for
SecA-mediated translocation (13). However, substitution of
Arg-357 does not interfere with SecA binding (11) but rather
prevents the SecA-dependent initiation of protein translocation (14).
Although many substrates of the translocon only require
the action of either SecA or the ribosome for translocation or
membrane insertion, the biogenesis of membrane proteins
with large periplasmic loops or domains, such as FtsQ and
CyoA, requires both functions (15, 16). Insight in the timing
and coordination of these activities is essential for the understanding of the mechanism of membrane protein insertion.
It has been suggested that non-translating ribosomes and
SecA do not compete for SecYEG binding and thus would be
able to bind simultaneously (17). However, the overlapping
binding sites (7, 8, 10) and the anticipated steric constraints
upon association of these two large ligands make this difficult to envision. Here, we have monitored the binding of
SecA, non-translating ribosomes and RNC complexes of the
monotopic membrane protein FtsQ to the membrane-embedded and detergent-solubilized SecYEG complex using
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During co-translational membrane insertion of membrane
proteins with large periplasmic domains, the bacterial SecYEG
complex needs to interact both with the ribosome and the SecA
ATPase. Although the binding sites for SecA and the ribosome
overlap, it has been suggested that these ligands can interact
simultaneously with SecYEG. We used surface plasmon resonance and fluorescence correlation spectroscopy to examine the
interaction of SecA and ribosomes with the SecYEG complex
present in membrane vesicles and the purified SecYEG complex
present in a detergent-solubilized state or reconstituted into
nanodiscs. Ribosome binding to the SecYEG complex is strongly
stimulated when the ribosomes are charged with nascent chains
of the monotopic membrane protein FtsQ. This binding is competed by an excess of SecA, indicating that binding of SecA and
ribosomes to SecYEG is mutually exclusive.
SecYEG Ligand Interaction
TABLE 1
Strains and plasmids used in this study
Strains/Plasmids
E. coli SF100
E. coli BL21(DE3) ⌬tig
pEK20
pNN260
pZW1
pEK20–148
pZW2
pZW3
pUC19Strep3FtsQSecM
pEK764
pEK765
pEK767
Characteristics
Source
F , ⌬lacX7, galE, galK, thi, rpsL, strA 4, ⌬phoA(pvuII), ⌬ompT
F ⫺, ompT, gal, dcm, lon, hsdSB(rB⫺ mB⫺) (DE3) ⌬tig
Cysteine-less SecYEG
Cysteine-less SecY(R357E)EG
Cysteine-less SecY(R255E, R256E)
SecY(L148C)EG
SecY(L148C, R255E, R256E)EG
SecY(L148C, R357E)EG
RNC-FtsQ108
RNC-FtsQ87
RNC-FtsQ77
RNC-FtsQ108:⌬TMS
(54)
(24)
(19)
(11)
This study
(27)
This study
This study
(20)
(21)
This study
This study
⫺
EXPERIMENTAL PROCEDURES
Strains and Plasmids—Inner membrane vesicles (IMVs)
with endogenous or overexpression levels of SecYEG were isolated from Escherichia coli SF100 transformed with the plasmids indicated in Table 1 as described previously (18). The
R357E mutation and R255E,R256E double mutation were
introduced into Cys-less SecY by site-directed mutagenesis
using pEK20 (19) as template, yielding pNN260 (11) and pZW1,
respectively. A unique cysteine at position 148 of SecY was
introduced into pZW1 and pNN260 yielding pZW2 and pZW3,
respectively.
Plasmid pUC19Strep3FtsQSecM (20) was used for the
isolation of FtsQ108 RNCs. For plasmids encoding FtsQ RNCs
of 77 or 87 residues and FtsQ with a deletion of the first transmembrane segment (TMS), the PstI-EcoRV fragment of
pUC19Strep3FtsQSecM (20) was exchanged with fragments
coding for FtsQ-(3– 41), FtsQ-(3–51), and FtsQ-(49 –122)
yielding pEK765, pEK764 (21), and pEK767, respectively. All
plasmids were verified by sequence analysis.
Ribosome and RNC Complex Purification—Non-translating
ribosomes were purified from E. coli MRE600 (22). Cells
were cultured in LB medium at 37 °C to an A660 of 0.6 and
harvested by centrifugation (9500 ⫻ g at 4 °C, 20 min). The
pellet was resuspended in buffer A (20 mM Tris-HCl, pH 7.5,
100 mM NH4Cl, 10.5 mM MgOAc, 0.5 mM EDTA, 1 mM
Tris(2-carboxyethyl)phosphine) and French-pressed twice
at 700 p.s.i. Cell debris was removed by centrifugation
(36,000 ⫻ g at 4 °C, 20 min) and the cleared lysate was laid on
top of a 1.1 M sucrose cushion in buffer A with 0.5 M NH4Cl
(buffer W) followed by centrifugation (72,000 ⫻ g, 4 °C,
19 h). The ribosomal pellet was washed with buffer W, and
the sucrose cushion step was repeated twice to yield higher
purity. The ribosome concentration was determined spectrophotometrically at a wavelength or 260 nm using an
extinction coefficient of 4.2 ⫻ 107 (23).
For purification of RNCs, E. coli BL21(DE3)⌬tig (24), which
lacks the trigger factor gene, was transformed with either
pUC19Strep3FtsQSecM, pEK764, pEK765, or pEK767 and
grown in LB supplemented with 100 ␮g/ml ampicillin at 30 °C
7886 JOURNAL OF BIOLOGICAL CHEMISTRY
to A600 of 0.5. After harvesting, cells were lysed according to
Evans et al. (25). In short, cells were resuspended in buffer R (50
mM Tris-HCl pH 7.5, 150 mM KCl, 10 mM MgCl2) and lysed by
two freeze-thaw cycles in the presence of 400 ␮g/ml lysozyme.
Cell debris was pelleted by centrifugation (twice at 30,000 ⫻ g,
4 °C, 30 min), and the cleared lysate was laid on a 1 M sucrose
cushion prepared in buffer R. After centrifugation (112,000 ⫻ g,
4 °C, 17 h), the ribosomal pellet was dissolved in buffer R and
loaded on a StrepTactin column (IBA). The column was
washed with two column volumes of buffer R containing 0.5 M
KCl and five column volumes of buffer R. RNCs were eluted
with two column volumes of buffer R containing 2.5 mM desthiobiotin. The eluate was concentrated using Millipore Amicon Ultra-4 or Ultra-15 centrifugal tubes (cut-off 50 kDa), and
the ribosome concentration was determined. The presence of
stalled nascent chains was confirmed by SDS-PAGE followed
by Western blotting using an antibody against the STREP-tag
(IBA).
Surface Plasmon Resonance (SPR)—SPR measurements were
performed on a Biacore 2000 system (GE Healthcare) as
described (26). In short, IMVs containing endogenous or
overexpressed levels of SecYEG (mutants) were immobilized
in the separate channels of a L1 sensor chip (GE Healthcare).
Buffer B (50 mM Tris-HCl, pH 7.5, 150 mM KCl, 5 mM MgCl2,
1 mM DTT, and 0.5 mg/ml BSA) containing SecA, ribosomes,
or RNCs was injected into the channels at a flow rate of 20
␮l/min, and SecYEG binding was probed at 25 °C. The binding surface of IMVs was regenerated by the injection of 100
mM Na2CO3, pH 10, followed by system equilibration using
buffer B. Data were corrected for background binding to the
IMVs containing endogenous levels of SecYEG. For SecAribosome competition experiments, ribosome binding was
measured in the presence or absence of a saturating concentration (96 nM or as indicated) of SecA in the running buffer
B. Data were fitted by nonlinear regression analysis of the
response levels at equilibrium using SigmaPlot (Systat Software Inc.). For the single site (A ⫹ B 7 AB) interaction
model including a non-saturable linear component the following equation was used,
Y⫽
B max ⫻ X
⫹ Ns ⫻ X
KD ⫹ X
(Eq. 1)
where Y is the SPR binding response at analyte (SecA or ribosome) concentration X, Bmax is the maximal binding response,
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two different methods, i.e. surface plasmon resonance and
fluorescence correlation spectroscopy. Our data demonstrate that SecA and the ribosome compete for binding to the
SecYEG complex and that substrate loading is an important
determining factor in this process.
SecYEG Ligand Interaction
KD is the dissociation constant, and Ns is the nonspecific binding coefficient.
For the two-site saturation model the following equation was
used.
B max1 ⫻ X Bmax2 ⫻ X
Y⫽
⫹
KD1 ⫹ X
KD2 ⫹ X
(Eq. 2)
冉
1
t
G共t兲 ⫽ ៮ 1 ⫹
␶D
N
冊
⫺1
冢
1
␻0 2 t
1⫹
z0 ␶D
冉 冊
冣
(Eq. 3)
៮i
where G(t) is the amplitude of the auto-correlation function, N
is the average number of fluorescent particles in the laser focus,
and ␶D is the diffusion time through the focus. To analyze
SecYEG diffusion in the presence of ribosomes and to estimate
fractions of free and ribosome-bound SecYEG, a two-component fitting model was applied,
MARCH 9, 2012 • VOLUME 287 • NUMBER 11
冊
⫺1
冢
冣
冢
冉
1
1
t
⫹ ៮ 1⫹
2
␶ D2
␻0 t
N2
1⫹
z 0 ␶ D1
冉 冊
⫻
1
␻0 2 t
1⫹
z 0 ␶ D2
冉 冊
冣
冊
⫺1
(Eq. 4)
៮ i and ␶Di are average number of fluorescent particles
where N
and diffusion time of ith species, respectively. The diffusion
times of SecYEG and SecYEG-ribosome complexes were preset
for the fitting procedure. For the free SecYEG the value was
directly measured, whereas the diffusion time of the SecYEGribosome complex was approximated with that of fluoresceinlabeled FtsQ108-RNC, which was experimentally determined
in our set-up (⬃900 ␮s).
SecA Co-sedimentation Assay—SecA (92 nM) was incubated
with non-translating ribosomes (60 nM), FtsQ108 (50 nM), and
FtsQ108⌬TMS (50 nM) in buffer B for 20 min at room temperature. Samples were loaded on a 35% (w/v) sucrose cushion and
centrifuged for 2 h at 350,000 ⫻ g, 4 °C. Pellets were dissolved in
SDS-PAGE sample buffer and analyzed by SDS-PAGE and silver staining.
In Vitro Protein Translocation Assay—In vitro translocation
of fluorescein-labeled proOmpA (C290S) was done as described (32). Translocated, protease-resistant proOmpA was
separated by SDS-PAGE and visualized with a LAS-4000
imager (FujiFilm) using the SyBr Blue Y515 Di filter.
Miscellaneous—SDS-PAGE, Western blotting, and silver
staining were performed according to standard protocols.
IMVs concentrations were determined using the Bio-Rad RC
DC protein assay kit using BSA as standard. ProOmpA(C290S)
and SecA were purified as described (33, 34). Quantification of
the SDS-PAGE bands was done using Aida/2D densitometry
(Raytest).
RESULTS
Detection of SecYEG-Ribosome Interaction by Surface Plasmon Resonance—We employed SPR to follow the binding of
ribosomes to the membrane-embedded SecYEG complex in
real time. This method was previously shown to accurately
detect the high affinity interaction between SecA and SecYEG
(11, 26). IMVs containing overexpressed levels of SecYEG were
immobilized on a Biacore L1 chip, and the association and dissociation of SecA (Fig. 1A) and ribosomes (Fig. 1B) was followed
in time. To correct for “bulk” contributions to the SPR signal
and nonspecific binding to the membrane and chip surface, all
measurements were corrected for binding to IMVs bearing
endogenous SecYEG levels that were immobilized in a reference channel (26) (supplemental Fig. S1A). Both SecA and ribosome injection resulted in a SecYEG-dependent SPR response
(Fig. 1B).
To validate that the observed SPR response reflected the
binding of ribosomes to the SecYEG complex, we analyzed the
interaction between ribosomes and two SecY mutant complexes, i.e. SecY(R255E,R256E)EG and SecY(R357E)EG. These
mutations are located in the SecY cytoplasmic loops C4 and C5,
respectively, and have been shown to disturb the SecYEG-riboJOURNAL OF BIOLOGICAL CHEMISTRY
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Fluorescence Correlation Spectroscopy (FCS)—For fluorescence microscopy, SecY(L148C)EG was overexpressed, purified, and labeled with AlexaFluor 488-C5-maleimide (Invitrogen) as described (27). Reconstitution of the fluorescently
labeled SecY(L148C)EG into MSP1D1 nanodiscs (28) was carried out as described (29, 30) with the following modifications.
A synthetic lipid composition consisting of 25 mol % dioleoylphosphatidylglycerol, 5 mol % cardiolipin, 30 mol % dioleoylphosphatidylethanolamine, and 40 mol % dioleoylphosphatidylcholine was used for nanodiscs formation, and lipids were
destabilized by 0.5% (v/v) Triton X-100 before SecYEG reconstitution. SecYEG-containing nanodiscs were isolated using a
Tricorn Superdex 200 10/300 column (GE Healthcare). The
concentration of reconstituted SecYEG-AlexaFluor 488 in the
collected fractions was determined based on the specific fluorophore absorbance.
FCS experiments were performed using the inverted confocal microscope LSM 710 equipped with the Confocor 3 module
(Carl Zeiss GmbH). A solution of 50 nM AlexaFluor 488 dye
with a known diffusion coefficient of 300 ⫻ 10⫺8 cm⫺2/s (31)
was used to adjust the laser intensity at 488 nm and to calibrate
the observation volume. Before the experiment SecYEG complexes were diluted to a concentration of ⬃100 nM in 50 mM
Tris-HCl, 100 mM NaCl, 10 mM MgCl2, and 5% (v/v) glycerol.
For detergent-solubilized SecYEG the buffer was also supplied
with 0.05% (w/v) n-dodecyl ␤-D-maltoside (DDM). Diffusiondriven fluctuations in the fluorescence intensity of SecYEGconjugated AlexaFluor 488 were recorded over 10 s, and the
measurements were repeated 10 times for each selected area.
Recorded traces were averaged. For each experimental condition at least 10 measurements were done at different positions
within the sample volume.
Autocorrelation curves were built and analyzed using ZEN
2010 software package (Carl Zeiss GmbH, Germany). Triplet
state population of the fluorophore was below 20%, and it was
omitted in the following analysis as the autocorrelation traces
were fitted within 10 ␮s to a 10-s time range. The data were
analyzed assuming free three-dimensional diffusion of SecYEG,
and the auto-correlation curves were fitted according to the
equation,
冉
1
t
G共t兲 ⫽ ៮ 1 ⫹
␶ D1
N1
SecYEG Ligand Interaction
FIGURE 2. SecA, ribosomes, and RNCs interact with the SecYEG complex with nanomolar affinity. SecYEG binding at increasing concentrations of SecA (A),
non-translating ribosomes (B), and FtsQ108 RNCs (C) was determined by SPR as described in Fig. 1. The equilibrium SPR responses were fitted by nonlinear
regression analysis. The fitting for SecA was performed based on a model assuming single site with a nonspecific binding parameter. For fitting for nontranslating ribosome and RNCs, a two-site saturation model was used. Binding data are summarized in Table 1.
some interaction in detergent solution (7). Both SecYEG
mutants were overexpressed to similar levels as Cys-less
SecYEG (supplemental Fig. S1A). The alteration in the charge
distribution in these cytosolic loops, however, resulted in a
reduced mobility of SecYEG on SDS-PAGE (supplemental Fig.
S1A). The SecY(R357E) mutation, which has been reported to
affect the initiation of SecA-dependent protein translocation
(13, 14), inhibited proOmpA translocation completely, whereas
the SecY(R255E,R256E)EG mutant was normally active (supplemental Fig. S1B). Importantly, both mutants allowed for significant SecA binding (Fig. 1A), indicating that the mutations
did not result in a major disturbance of the loop conformation.
However, the SPR responses upon injection of ribosomes over
immobilized SecY(R255E, R256E)EG or SecY(R357E)EG IMVs
were dramatically reduced as compared with Cys-less SecYEG
IMVs (Fig. 1B). These observations are consistent with the
notion that the mutated loop residues are important for
the SecYEG-ribosome interaction (7) and demonstrate that the
SPR method genuinely monitors the interaction between ribosomes and the membrane-embedded SecYEG complex.
Previously, we observed that the SecA association and dissociation phases do not fit to a simple bimolecular interaction
7888 JOURNAL OF BIOLOGICAL CHEMISTRY
TABLE 2
Dissociation constants and apparent binding levels for the interaction
of SecA, ribosomes, or FtsQ108 RNCs with the SecYEG complex as
determined by surface plasmon resonance
R2 ⫽ coefficient of determination; KD1 and KD2 are the dissociation constants 1 and
2, respectively. Bmax1 and Bmax2 are the maximum obtained binding values associated with the two dissociation constants. Ns, nonspecific binding. RU, response
units.
Analyte
SecA
Ribosomes
FtsQ108RNCs
Bmax1
Bmax2
RU
RU
85
27
117
70
114
KD1
K2
nM
nM
4.5
0.03
0.12
8.8
7.9
Ns
R2
0.15
0.9931
0.9978
0.9973
model (26, 35). Similarly, an accurate determination of the association and dissociation rates of ribosome binding could not be
obtained by such simple data fitting models. Therefore, an
apparent affinity of the interaction was determined by nonlinear regression analysis of the response levels at equilibrium (Fig.
2). For the SecYEG-SecA interaction, this resulted in an apparent dissociation constant (KD) of 4.5 nM when fitted according
to a simple single site (A ⫹ B 7 AB) interaction model including a non-saturable linear component (Fig. 2A, Table 2). This
value is similar to those obtained previously by SPR, fluoresVOLUME 287 • NUMBER 11 • MARCH 9, 2012
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FIGURE 1. Specific binding of SecA and ribosomes to the SecYEG complex monitored by SPR. Shown is an SPR sensogram of the binding of SecA (A) or
non-translating ribosomes (B) to IMVs containing overexpressed levels of Cys-less SecYEG (solid), SecY(R357E)EG (spaced dashed), or SecY(R255E,R256E)EG
(dashed). Binding was measured at 25 °C with a flow rate of 20 ␮l/min. Data were corrected for background binding to IMVs containing endogenous SecYEG
levels. The ligand association and dissociation phases are represented above the sensograms by black and white bars, respectively. The concentration of SecA
and ribosomes was 48 and 27 nM, respectively.
SecYEG Ligand Interaction
cence microscopy, and biochemical methods (11, 26, 33, 36). In
contrast, the ribosome binding responses fitted best to a twosite saturation model yielding apparent KD values of 0.03 and
8.8 nM, respectively (Fig. 2B, Table 2). Although the fit does not
explain the mechanism of the interaction, the lower affinity
value is in the same order as reported previously by equilibrium
binding experiments, i.e. 14 –17 nM with IMVs (17) and 6 nM
with SecYEG proteoliposomes (37). These results demonstrate
that both SecA and the ribosome interact with the SecYEG
complex with nanomolar affinity.
Increased Ribosome Binding in Presence of Nascent Chain—
During membrane protein insertion, the SecYEG complex
interacts with translating ribosomes. To determine whether the
presence of a nascent chain influences the SecYEG-ribosome
interaction, ribosomes carrying chimeric nascent chains of the
E. coli inner membrane protein FtsQ were isolated. To halt
translation, constructs were used in which the stalling motif of
the E. coli secretion monitor protein SecM (38) was fused
behind the first 41, 51, or 72 amino acids of FtsQ, resulting in
nascent chains of 77, 87, or 108 amino acids long (FtsQ77,
FtsQ87, FtsQ108 (20), respectively). At these nascent chain
lengths the FtsQ TMS is expected to be either half-exposed
(FtsQ77), nearly completely exposed (FtsQ87), and completely
exposed (FtsQ108) from the ribosomal exit site (4) (Fig. 3A).
Constructs were preceded by a triple STREP-tag to allow separation of RNCs from non-translating ribosomes (20). After isolation, the presence of the nascent chains was verified by
immunoblotting against an STREP-tag antibody (Fig. 3B). Surprisingly, injection of the FtsQ87 and FtsQ108 RNCs resulted in
MARCH 9, 2012 • VOLUME 287 • NUMBER 11
an SPR response that was 3–5-fold higher compared with nontranslating ribosomes (Fig. 3C), indicating that the SecYEGribosome interaction is strongly stimulated by the presence of a
nascent chain. The shorter FtsQ77 RNC increased binding to a
lesser extent (Fig. 3C), suggesting that the enhanced interaction
of translating ribosomes with the SecYEG complex is dependent on the length of the nascent chain with an optimal interaction when the TMS of FtsQ is exposed from the ribosome
tunnel. Indeed, injection of a FtsQ nascent chain of 108 residues
without TMS (FtsQ108-⌬TMS) yielded a significantly reduced
binding response compared with FtsQ108 exposing the TMS
(Fig. 3D), suggesting that TMS contributes to the enhanced
binding of RNCs to the translocon.
The interaction between RNCs and SecYEG was further
studied using the FtsQ108 construct that has previously also
been used for cross-linking and structural studies (4, 6). As
observed for non-translating ribosomes, the R255E,R256E
mutations in the C4 loop of SecY abrogated the interaction with
FtsQ108 RNCs (Fig. 3E, dashed line). However, these RNCs
showed substantial interaction with SecY(R357E)EG (Fig. 3E,
spaced dashed line), which is consistent with our previous data
that indicated that the R357E mutation still supports membrane protein insertion (14). These data demonstrate that
translating and non-translating ribosomes interact differently
with the SecYEG complex.
Interaction of Purified SecYEG Complex with SecA and
Ribosomes—To verify the effect of a nascent chain on the
SecYEG-ribosome interactions, we monitored the binding
reaction between the purified components. Binding was probed
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FIGURE 3. The presence of a nascent chain enhances the ribosome-SecYEG interaction. A, shown is a schematic illustration of the FtsQ RNCs with various
chain lengths. B, shown is Western blotting of RNCs of increasing chain length pretreated with 100 mM NaOH. To overcome poor transfer efficiency, a double
amount of the FtsQ77 RNC was loaded. C, shown is binding of 27 nM ribosomes (dashed black) or RNCs with a FtsQ nascent chain length of 77 (solid gray), 87
(solid black), and 108 (dash gray) residues to immobilized IMVs containing overexpressed Cys-less SecYEG. D, binding of 13.5 nM non-translating ribosomes
(black dashed), FtsQ108 RNCs (solid line), and FtsQ108-⌬TMS RNCs (gray dashed) is shown. E, binding of 27 nM FtsQ108 RNCs to immobilized IMVs containing
overexpressed Cys-less SecYEG (solid line), SecY(R357E)EG (spaced dashed), and SecY(R255E,R256E)EG (dashed) is shown.
SecYEG Ligand Interaction
using FCS, a sensitive confocal microscopy technique that
allows for the analysis of molecular interactions in equilibrium
at nanomolar substrate concentrations (39). In FCS, fluctuations of fluorescence resulting from fluorescently labeled proteins diffusing through the femtoliter-sized confocal excitation
volume are monitored and auto-correlated over the measurement time. Temporal decay in the correlation function provides a precise estimate of the diffusional mobility of the fluorescently labeled biomolecule. Binding of the fluorescent
protein to large non-labeled components can be detected as
alterations of its mobility (Fig. 4A). First, the interaction
between the solubilized SecYEG complex and ribosomes was
determined in a solution containing 0.05% of the detergent
DDM. Purified and fluorescently labeled SecY(C148)EG-AlexaFluor 488 (⬃100 nM) was illuminated when diffusing through
the laser focal volume, and temporary fluctuations in the fluorescence intensity were recorded and used to build an autocorrelation curve (Fig. 4A). The FCS data were analyzed assuming
unrestricted three-dimensional diffusion of the labeled SecYEG
in solution, and diffusion coefficient (D) of 26 ⫾ 3 ⫻ 10⫺8
cm⫺2/s was obtained. The addition of an excess SecA (1.8 ␮M)
decreased the diffusion coefficient of solubilized SecYEG only
slightly by ⬃10% to 23.9 ⫾ 1.5 ⫻ 10⫺8 cm⫺2/s (data not shown).
However, upon the addition of an excess FtsQ108 RNCs (250
nM), the mobility of SecY(L148C)EG-AlexaFluor 488 reduced
substantially, resulting in a pronounced shift of the autocorrelation trace (Fig. 4A). The average diffusion time decreased
⬃3-fold, and the D reduced to 10.0 ⫾ 0.9 cm⫺2/s, suggesting
that SecYEG was bound to RNC and diffused slowly as a part of
the large complex. In agreement with the Einstein-Stokes equation, the observed reduction in the diffusion coefficient correlated with the 3-fold difference in dimensions of SecYEG protein enlarged by a belt of detergent molecules (radius R ⬃ 3 nm
(40, 41) and the ribosome (R ⬃ 10 nm (42)). The addition of an
7890 JOURNAL OF BIOLOGICAL CHEMISTRY
excess (250 nM) of non-translating ribosomes to SecYEG
reduced the translocon mobility to a lesser extent (Fig. 4A),
suggesting that a large fraction of the SecYEG was not bound to
these ribosomes and retained their high diffusional mobility. To
quantify the binding efficiency, the autocorrelation traces of
SecYEG were fitted assuming two SecYEG fractions in the
ensemble: uncomplexed and ribosome/RNC-bound. The data
suggested that more than 98% of SecYEG was bound with
FtsQ108 RNCs, whereas only 67% was bound to non-translating ribosomes (Fig. 4B). As was reported previously (7), the
R255E,R256E and R357E mutations in SecYEG reduced binding of non-translating ribosomes in the detergent environment
(supplemental Fig. S2). However, in agreement with the SPR
data presented above, the binding defect of the R357E mutant
could be partially restored by the presence of a nascent chain
(supplemental Fig. S2).
Because the above studies were performed in detergent
solution, we also investigated the role of the lipids in SecA and
RNC binding to SecYEG. Herein, fluorescently labeled
SecY(C148)EG-AlexaFluor 488 was reconstituted into small
lipid patches, known as nanodiscs (28). These provide a physiologically relevant lipid environment for the embedded membrane proteins (supplemental Fig. S3). Because nanodiscs are
monodisperse in aqueous solution, they are well suited for
fluorescence microscopy applications, including FCS. The diffusion coefficient of SecYEG reconstituted into nanodiscs
(SecYEG-Nd) was similar to that of detergent-solubilized
SecYEG (D ⫽ 27 ⫾ 3 ⫻ 10⫺8 cm⫺2/s). In agreement with previous reports (8), no binding was observed for non-translating
ribosomes. In contrast, the mobility of SecYEG-Nd decreased
substantially in the presence of FtsQ108 RNCs, indicating that
the presence of a nascent chain triggered ribosome binding to
the membrane-embedded translocon. Fitting the autocorrelation curves with the two-component model suggested that 82%
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FIGURE 4. The SecYEG-ribosome interaction is stimulated by the presence of a nascent chain and the lipid environment. A, fluorescence autocorrelation
traces describe diffusion properties of DDM-solubilized SecYEG-AlexaFluor 488. Binding of ribosomes/RNCs reduces SecYEG mobility and causes the traces to
shift toward longer diffusion times. B, when assayed in the presence of the detergent 0.05% (w/v) DDM, non-translating ribosomes bind SecYEG less efficiently
than RNCs. This effect is further enhanced for SecYEG reconstituted into the lipid environment of nanodiscs.
SecYEG Ligand Interaction
of the SecYEG formed complexes with RNCs (Fig. 4B). As
observed with the native membrane-embedded and solubilized
SecYEG complexes, the interaction of FtsQ108 with SecYEG-Nd
was partially inhibited by the R357E mutation and more
strongly blocked by the R255E,R256E mutation of SecYEG
(supplemental Fig. S2). Together, these FCS results corroborate
the observation made by SPR that the presence of the nascent
chain strongly promotes the SecYEG-ribosome interaction the
SPR data but also indicate that the interaction between SecYEG
and the ribosome is influenced by the environment of the
SecYEG complex (solubilized versus membrane-embedded)
showing a greater interaction between empty ribosomes and
SecYEG when present in detergent.
Competitive Binding of SecA and Ribosomes—It has been
proposed that SecA and ribosomes interact with the translocon
non-competitively (17). This would imply that ribosomes and
SecA bind to different and independent sites on SecYEG. To
investigate this phenomenon, we designed an SPR competition
experiment to determine whether the SecYEG complex can
accommodate both SecA and the ribosome simultaneously.
First, the SecYEG complexes in the immobilized IMVs were
saturated with SecA using a running buffer containing excess
SecA (96 nM). Subsequent injection of ribosomes in the SecAcontaining running buffer resulted in a binding response that
MARCH 9, 2012 • VOLUME 287 • NUMBER 11
was 2–3-fold lower than in the absence of SecA (Fig. 5, A and B).
Stabilizing the SecYEG-SecA interaction by the addition of
AMP-PNP, which prevents the dissociation of SecA from the
SecYEG complex (26, 43), or a combination of ATP and azide,
which prevents the release of SecA from SecYEG (44), further
decreased the ribosome binding up to 8-fold (Fig. 5C). As a
control, AMP-PNP or ATP-azide alone only slightly affected
the SecYEG-ribosome interaction (Fig. 5C). When the ribosomes were loaded with the FtsQ108 nascent chain, the
SecYEG interaction was affected even more severely by the
presence of SecA (Fig. 5D). SecA has been reported to interact
with ribosomes and RNCs (45, 46). However, the reduced interaction was not caused by sequestration of ribosomes or RNCs
by the SecA present in the running buffer, as co-sedimentation
assays showed that under the conditions used for SPR less than
3% of the RNCs were SecA-bound (Fig. 5E). This is consistent
with the observation that the SecA-ribosome interaction is of
low affinity (0.9 ␮M) (45). The results, therefore, indicate that
SecA competes with both translating and non-translating ribosomes for SecYEG binding.
Detergents Affect Interaction between SecYEG and Ligands
SecA and Ribosomes—Because the diffusion coefficient of
SecYEG was strongly affected by binding of a ribosome, but not
by SecA (see above), the RNC- and SecA-bound SecYEG popJOURNAL OF BIOLOGICAL CHEMISTRY
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FIGURE 5. SecA competes with the ribosome for SecYEG binding. A, equilibrium SPR responses at increasing concentrations of non-translating ribosomes
were measured in the presence (white) or absence (black) of SecA (96 nM) in the SPR running buffer. B, binding of non-translating ribosomes (6.75 nM) in the
presence or absence of SecA (96 nM) in shown. C, normalized ribosome binding in the presence (white) or absence (black) of 96 nM SecA with or without
AMP-PNP (1 mM) or ATP (1 mM) and azide (5 mM) is shown. Binding responses were normalized to the equilibrium response in the absence of SecA. D, binding
of FtsQ108 RNCs (3.38 nM) in the presence or absence of a SecA (96 nM) is shown. E, co-sedimentation of SecA (92 nM) with ribosomes (60 nM), FtsQ108 (50 nM),
and FtsQ108; ⌬TMS (50 nM) is shown. The SecA input represents amount of SecA added to the reaction mixture. RU, response units.
SecYEG Ligand Interaction
ulations can be discriminated based on their diffusional mobility. SecA and FtsQ108 RNC were added to DDM-solubilized
SecY(L148C)EG-AlexaFluor 488 together, and the translocon
diffusion was monitored by FCS. AMP-PNP was added to stabilize the SecYEG-SecA complex. Assuming that both SecYEGSecA and SecYEG-RNC complexes are formed within the
ensemble, a two-component model can be used for analysis.
Surprisingly, even in the presence of a large excess of SecA (1.8
␮M versus 250 nM RNCs), 87% of the translocons were complexed with the RNCs in detergent solution (Fig. 6). To determine whether the detergent environment of the SecYEG complex caused this apparent discrepancy with the SPR results, the
same experiment was performed using the nanodisc-reconstituted SecYEG complex. The SecYEG-Nd-SecA complex stabilized by AMP-PNP manifested a D of 23 ⫾ 3 ⫻ 10⫺8 cm⫺2/s,
which is similar to SecYEG-Nd alone. This allows for a discrimination between SecA and RNC FtsQ108 binding to
SecYEG-Nd based on the translocon mobility only. When both
SecA and FtsQ108 RNCs were added to SecY(L148C)EG-Nd in
the presence of 1 mM AMP-PNP, a clear competition for
SecYEG binding was observed, and the propensity to form a
complex with the RNCs decreased with the SecA-RNC ratio
(Fig. 6). In the presence of an elevated AMP-PNP concentration
(5 mM), RNC binding was further reduced, up to 3-fold (supplemental Fig. S4). These results indicate that the interaction
between SecYEG, translating ribosomes, and SecA is strongly
dependent on the molecular environment; although detergents
promote ribosome binding and weaken the competitive interaction with SecA, SecA and translating ribosomes bind competitively to the membrane-embedded SecYEG. Importantly,
the FCS data corroborate with the SPR data and demonstrate
that SecA and (non)translating ribosomes compete for binding
to the membrane-embedded SecYEG complex.
DISCUSSION
During the biogenesis of membrane proteins with large
periplasmic domains, both the ribosome and SecA need to
associate with the SecYEG complex to drive the membrane par-
7892 JOURNAL OF BIOLOGICAL CHEMISTRY
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FIGURE 6. SecA competes with ribosomes for binding to SecYEG reconstituted into nanodiscs. Binding of RNCs to purified SecYEG in nanodiscs in the
presence of increasing concentrations of SecA with AMP-PNP. The fraction of
RNC-bound SecYEG complex was determined by FCS as described under
“Results.” Detergent-solubilized SecYEG primarily interacted with RNC even
in a large excess of SecA (white bar).
titioning of hydrophobic segments and the translocation of
hydrophilic domains, respectively. How the translocon is able
to coordinate the binding of these two ligands is not well understood. Here we have used two distinct spectroscopic techniques
to follow the binding of SecA and of translating and non-translating ribosomes to the SecYEG complex.
SPR and FCS readily detected the association of ribosomes
with the SecYEG complex present in membrane vesicles and
the purified SecYEG present in detergent solution or reconstituted into nanodiscs. Although the molecular environment
influences the observed interactions, the interactions are specific and inhibited by mutations in SecY that were previously
reported to result in ribosome binding defects. Interestingly,
ribosome binding to the SecYEG complex was highly stimulated by the presence of a nascent chain, in particular when this
nascent chain exposed a hydrophobic polypeptide sequence.
The presence of a nascent chain also partially relieved the ribosome binding defect of the SecY(R357E) mutant. Taken
together this suggests that translating and non-translating ribosomes interact differently with the SecYEG complex and indicates an interplay, possibly a cooperative effect, of the nascent
chain in the SecYEG-ribosome interaction. A recent cryo-EM
structure of an FtsQ RNC complex with the translocon indicated that the nascent chain engages in interactions between
the ribosomal proteins L23 and L24 and the loops of SecY,
resulting in conformational changes in the participating loops
(8). These interactions and conformational changes might
tighten the interaction between the RNC complex and the
translocon, and this may be promoted by hydrophobic
stretches in the nascent polypeptide chain.
The interaction between translating or non-translating ribosomes and the membrane-embedded SecYEG complex was
competed by SecA, demonstrating that the two ligands cannot
bind the translocon simultaneously. Surprisingly, even though
the presence of a nascent chain enhances the ribosome-translocon interaction, SecA competed stronger with translating than
with non-translating ribosomes. Because our current understanding of the exact mechanism of the translocon-RNC interaction is still limited, this observation is not readily explained.
However, it is clear from the SPR data that the interaction is not
a simple one-to-one binding event. Using nonlinear regression
analysis of the response levels at equilibrium, ribosome binding
data could only be fitted to a “two-site saturation” model
according to which SecYEG partly interacted with the ribosomes with an apparent affinity in the low nanomolar scale
consistent with previously reported values (17, 37) and partly
with a very high affinity that was particularly prominent upon
binding of RNCs. This two-site saturation model suggests that
the dramatically increased binding response in the presence of
a nascent chain is caused by an increase in the number of binding sites rather than an improved affinity. Possibly, RNCs
recruit binding sites that were previously masked. Alternatively, binding of a RNC to a SecYEG complex causes an intrinsically higher SPR signal than binding of a non-translating ribosome, whereas the number of binding sites remains constant.
This could for instance occur when the nascent chain, by interacting with SecYEG, pulls the ribosome closer to the membrane
surface, which in turn would result in an increased SPR signal.
SecYEG Ligand Interaction
In this scenario the interaction with RNCs would direct the
SecYEG complex toward the high affinity state (see Table 2:
50% high affinity sites for RNCs versus 25% for non-translating
ribosomes). This very tight binding of translating ribosomes to
the translocon could be the result of multiple steps, so that
inhibition of RNC binding by SecA could occur if the initial
association is of relatively low affinity. Further studies will be
required to elucidate the molecular basis of the ribosome
translocon interaction.
SecA-ribosome competition only occurred when SecYEG
was in its native, membrane-embedded state, as the presence of
SecA did not affect the SecYEG-ribosome interaction in detergent solution. Because FCS indicated that solubilized SecYEG
has a higher propensity to bind non-translating ribosomes
compared with the membrane-embedded SecYEG (this study)
and detergents have been shown to affect the activity and oligomeric state of SecA (47– 49), it is likely that the interaction
between SecA, the ribosome, and the solubilized SecYEG complex is affected by the presence of the detergent. The observation that the molecular environment is a critical factor in the
interaction between the translocon and its soluble interactions
partners should be considered in future studies.
The observation that SecA and the ribosome compete for
binding to the SecYEG complex even when the ribosome is
charged with a nascent chain of a SecA-dependent membrane
protein implies that during membrane protein insertion, SecA
and the ribosome do not interact with the translocon simultaneously. Because many membrane proteins contain large
periplasmic domains, the competitive interaction of SecA and
the ribosome has implications for the membrane insertion
mechanism. Although for single-spanning membrane proteins
like FtsQ it is possible to envision a scenario where the ribosome dissociates from the SecYEG complex after membrane
partitioning of the TMS to allow SecA binding, the sequence of
events will be more complicated when a large periplasmic
domain is followed by another TMS. For these proteins, SecA
and the ribosomes might need to cycle on and off from the
MARCH 9, 2012 • VOLUME 287 • NUMBER 11
translocon in a sequential manner, a process for which the timing needs to be carefully orchestrated. It is plausible that the
SecYEG affinity to SecA and translating ribosomes depends on
the hydrophobicity of the emerging polypeptide chain. The
ribosome might be released from the SecYEG complex once it
encounters a large enough polar periplasmic polypeptide
domain that would allow SecA to bind to this domain followed
or concomitantly with SecYEG binding (Fig. 7). The micromolar SecA concentrations in the cytoplasm would ensure efficient cycling of SecA on SecYEG to translocate these polar
domains (33). Likewise, SecA may dissociate from the SecYEG
complex once it encounters a newly emerging TMS. In this
respect, SecA has been reported to dissociate from the SecYEG
complex when it encounters a potential TMS during the translocation of a secretory protein (50, 51). Early recognition of a
TMS by the translating ribosome (52) may restore the high
affinity interaction between SecYEG and the ribosome for the
insertion of the next TMS. Future studies should be directed to
unravel the interplay between SecA, RNCs, and the SecYEG
complex including targeting factors such as signal recognition
particle and FtsY (53).
Acknowledgments—We thank Ilja Küsters, Jelger Lycklama á Nijeholt, and Marko Sustarsic for valuable discussion and technical
assistance and Natalia Dudkina for cryoelectron microscopy experiments. We thank Nenad Ban (ETH, Zürich, Switzerland) and
Bernd Bukau (ZMBH, Heidelbeg, Germany) for kind gifts of
pUC19Strep3FtsQSecM and E. coli BL21(DE3)⌬tig::kan, respectively.
We thank Stephen Sligar (University of Illinois, Urbana, IL) for the
plasmids expressing the MSP protein.
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